Methods for controlling the galactosylation profile of recombinantly-expressed proteins

ABSTRACT

The present invention relates to methods for modulating the glycosylation profile of recombinantly-expressed proteins. In particular, the present invention relates to methods of controlling the galactosylation profile of recombinantly-expressed proteins by supplementing production medium, e.g., a hydrolysate-based or a chemically defined medium, with manganese and/or D-galactose

RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 15/014,694, filed on Feb. 3, 2016; which is adivisional application of U.S. patent application Ser. No. 14/619,799,filed on Feb.11, 2015, now U.S. Pat. No. 9,255,143, issued on Feb. 9,2016; which is a continuation application of U.S. patent applicationSer. No. 14/493,068, filed on Sep. 22, 2014, now U.S. Pat. No.9,090,688, issued on Jul. 28, 2015; which is a divisional application ofU.S. patent application Ser. No. 13/457,020, filed on Apr. 26, 2012, nowU.S. Pat. No. 9,062,106, issued on Jun. 23, 2015; which claims thebenefit of U.S. Provisional Application Ser. No. 61/479,727, filed onApr. 27, 2011. The entire contents of each of the foregoing applicationsare incorporated herein by reference.

1. INTRODUCTION

The present invention relates to methods for modulating theglycosylation profile of recombinantly-expressed proteins. Inparticular, the present invention relates to methods of controlling thegalactosylation profile of recombinantly-expressed proteins bysupplementing production media with manganese and/or galactose.

2. BACKGROUND OF THE INVENTION

Utilization of a particular type of production media, e.g.,hydrolysate-based media or chemically defined media (“CD” or “CDM”), forCHO cell cultures producing recombinant proteins can enhance cell growthand target protein production. However, recombinant proteins produced indifferent CD or hydrolysate-based media can exhibit large differences intheir product quality profile. In certain instances, this variabilitycan lead to increases in the fraction of the agalactosyl fucosylatedbiantennary oligosaccharides NGA2F+NGA2F−GlcNAc and decreases in thefraction of galactose-containing fucosylated biantennaryoligosaccharides NA1F+NA2F. Shifts in the glycosylation profile ofrecombinant proteins of this magnitude are significant as these shiftsmay render the resulting production lots of the target protein out ofcompliance with approved process specifications.

3. SUMMARY OF THE INVENTION

The present invention relates to methods for modulating theglycosylation profile of recombinantly-expressed proteins. Inparticular, the present invention relates to methods of controlling thegalactosylation profile of recombinantly-expressed proteins bysupplementing production media with manganese and/or galactose. Incertain embodiments the production media is a hydrolysate-based media ora CD media.

In certain embodiments, the present invention is directed to methods ofcontrolling the galactosylation profile of recombinantly-expressedantibody. In certain embodiments, the recombinantly-expressed antibodyis an anti-TNFα antibody. In certain embodiments, therecombinantly-expressed anti-TNFα antibody is adalimumab.

In certain embodiments, the present invention is directed to methods ofcontrolling the galactosylation profile of recombinantly-expressedproteins by supplementing a production medium, e.g., a hydrolysate-basedor a CD medium, used in the production of recombinantly-expressedproteins with manganese and/or galactose. In certain embodiments, themanganese supplement can take the form of any biologically-acceptablemanganese salt, for example, but not limited to, manganese (II)chloride. In certain embodiments, the galactose supplement can take theform of any biologically-acceptable galactose-containing compound, forexample, but not limited to, D-(+)-galactose.

In certain embodiments, the present invention is directed to methods ofcontrolling the galactosylation profile of recombinantly-expressedproteins by supplementing a production medium, e.g., a hydrolysate-basedor a CD medium, used in the production of recombinantly-expressedproteins with a sufficient amount of manganese and/or amanganese-containing supplement to achieve the following manganeseconcentrations in the production media: at least about 0.1, at leastabout 0.2, at least about 0.5, at least about 1.0, at least about 10, atleast about 20, at least about 25, at least about 40, at least about 50,at least about 60, at least about 75, at least about 80, or at leastabout 100 μM, wherein that production media is used to dilute asupplement-free cell culture growth media containing no supplement by aratio of about 1:4 or about 1:5 (supplement-free growthmedia:supplemented production media). In certain embodiments, thepresent invention is directed to methods of controlling thegalactosylation profile of recombinantly-expressed proteins bysupplementing a production medium, e.g., a hydrolysate-based or a CDmedium, used in the production of the recombinantly-expressed proteinswith sufficient galactose and/or galactose-containing supplement toachieve the following galactose concentrations in the production media:at least about 1, at least about 4, at least about 5, at least about 10,at least about 15, at least about 20, at least about 30, at least about40, at least about 60, or at least about 100 mM, wherein that productionmedia is used to dilute a supplement-free cell culture growth mediacontaining no supplement by a ratio of about 1:4 or about 1:5(supplement-free growth media:supplemented production media).

In certain embodiments, the present invention is directed to methods ofcontrolling the galactosylation profile of recombinantly-expressedproteins by supplementing a production medium, e.g., a hydrolysate-basedor a CD medium, used in the production of recombinantly-expressedproteins with sufficient manganese and/or a manganese-containingsupplement and sufficient galactose and/or galactose-containingsupplement to achieve at least about the following manganese (Mn) andgalactose (Gal) concentrations in the production media presented as Mn(μM)/Gal (mM): 0/1, 0/4, 0/5, 0/10, 0/15, 0/20, 0/30, 0/40, 0/60, 0/100,0.1/0, 0.2/0, 0.5/0, 1.0/0, 10/0, 20/0, 25/0, 40/0, 50/0, 75/0, 80/0,100/0, 0.2/1, 0.2/4, 0.2/30, 0.5/1, 0.5/4, 0.5/30, 10/10, 10/20, 10/40,20/10, 20/20, 20/40, 25/15, 40/10, 40/20, 40/40, 40/100, 50/30, 60/20,60/40, 60/100, 80/20, 80/40, 80/100, 100/20, 100/40, 100/100, whereinthat production media is used to dilute a supplement-free cell culturegrowth media containing no supplement by a ratio of about 1:4 or about1:5 (supplement-free growth media:supplemented production media).

In certain embodiments, the present invention is directed to methods ofcontrolling the galactosylation profile of recombinantly-expressedproteins by supplementing a production medium, e.g., a hydrolysate-basedor a CD medium, used in the production of recombinantly-expressedproteins with sufficient manganese and/or a manganese-containingsupplement and sufficient galactose and/or galactose-containingsupplement to achieve at least about the following manganese (Mn) andgalactose (Gal) concentrations in the production media presented as Mn(μM)/Gal (mM): 0.2/1, 0.2/4, 0.2/30, 0.5/1, 0.5/4, 0.5/30, 10/10, 10/20,10/40, 20/10, 20/20, 20/40, 25/15, 40/10, 40/20, 40/40, 40/100, 50/30,60/20, 60/40, 60/100, 80/20, 80/40, 80/100, 100/20, 100/40, 100/100,wherein that production media is used to dilute a supplement-free cellculture growth media containing no supplement by a ratio of about 1:4 orabout 1:5 (supplement-free growth media: supplemented production media).

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A depicts the culture growth of adalimumab-producing CHO cell linein CDM GIA-1 in batch shake flasks. FIG. 1B depicts the viability ofadalimumab-producing CHO cell line in CDM GIA-1 in batch shake flasks.FIG. 1C depicts the normalized titer of adalimumab-producing CHO cellline in CDM GIA-1 in batch shake flasks.

FIG. 2A depicts the culture growth of adalimumab-producing CHO cell linein CDM GIA-1 in fed-batch 3 L bioreactors. FIG. 2B depicts the viabilityof adalimumab-producing CHO cell line in CDM GIA-1 in fed-batch 3 Lbioreactors. FIG. 2C depicts the normalized titer ofadalimumab-producing CHO cell line in CDM GIA-1 in fed-batch 3 Lbioreactors.

FIG. 3A depicts the galactosylation profile (NGA2F+NGA2F-GlcNac) ofadalimumab in CHO cell line in CDM GIA-1 in batch shake flasks. FIG. 3Bdepicts the galactosylation profile (NA1F+NA2F) of adalimumab in CHOcell line in CDM GIA-1 in batch shake flasks.

FIG. 4 depicts the percentage galactosylation change of adalimumab inCDM GIA-1 in batch shake flasks relative to control.

FIG. 5 summarizes the effect of manganese and/or galactose addition toCDM GIA-1 on galactosylation of adalimumab relative to control in CHOcell line.

FIG. 6A depicts the galactosylation profile (NGA2F+NGA2F-GlcNac) ofadalimumab in CHO cell line in CDM GIA-1 in fed-batch 3 L bioreactors.FIG. 6B depicts the galactosylation profile (NA1F+NA2F) of adalimumab inCHO cell line in CDM GIA-1 in fed-batch 3 L bioreactors.

FIG. 7A depicts the culture growth of CHO cell line in CDM HyCloneCDM4CHO in batch shake flasks. FIG. 7B depicts the viability of CHO cellline in CDM HyClone CDM4CHO in batch shake flasks.

FIG. 8A depicts the galactosylation profile (NGA2F+NGA2F-GlcNac) ofadalimumab in CHO cell line in CDM HyClone CDM4CHO in batch shakeflasks. FIG. 8B depicts the galactosylation profile (NA1F+NA2F) ofadalimumab in CHO cell line in CDM HyClone CDM4CHO in batch shakeflasks.

FIG. 9 summarizes the effect of manganese and/or galactose addition toCDM HyClone CDM4CHO on galactosylation of adalimumab relative to controlin CHO cell line.

FIG. 10A depicts the culture growth of CHO cell line in hydrolysatemedia in batch shake flasks. FIG. 10B depicts the viability of CHO cellline in hydrolysate media in batch shake flasks.

FIG. 11A depicts the galactosylation profile (NGA2F+NGA2F-GlcNac) ofadalimumab in CHO cell line in hydrolysate media in batch shake flasks.FIG. 11B depicts the galactosylation profile (NA1F+NA2F) of adalimumabin CHO cell line in hydrolysate media in batch shake flasks.

FIG. 12 summarizes the effect of manganese and/or galactose addition tohydrolysate media on galactosylation of adalimumab relative to controlin CHO cell line.

FIG. 13A depicts the culture growth of adalimumab-producing CHO cellline #2 in CDM GIA-1 in batch shake flasks. FIG. 13B depicts theviability of adalimumab-producing CHO cell line #2 in CDM GIA-1 in batchshake flasks.

FIG. 14A depicts the galactosylation profile (NGA2F+NGA2F-GlcNac) ofadalimumab in CHO cell line #2 in CDM GIA-1 in batch shake flasks. FIG.14B depicts the galactosylation profile (NA1F+NA2F) of adalimumab in CHOcell line #2 in CDM GIA-1 in batch shake flasks.

FIG. 15 summarizes the effect of manganese and/or galactose addition toCDM GIA-1 on galactosylation of adalimumab relative to control in CHOcell line #2.

FIG. 16A depicts the culture growth of adalimumab-producing CHO cellline #3 in CDM GIA-1 in fed-batch 3 L bioreactors. FIG. 16B depicts theviability of adalimumab-producing CHO cell line #3 in CDM GIA-1 infed-batch 3 L bioreactors. FIG. 16C depicts the normalized titer ofadalimumab-producing CHO cell line #3 in CDM GIA-1 in fed-batch 3 Lbioreactors.

FIG. 17A depicts the galactosylation profile (NGA2F+NGA2F-GlcNac) ofadalimumab in CHO cell line #3 in CDM GIA-1 in fed-batch 3 Lbioreactors. FIG. 17B depicts the galactosylation profile (NA1F+NA2F) ofadalimumab in CHO cell line #3 in CDM GIA-1 in fed-batch 3 Lbioreactors.

FIG. 18 summarizes the effect of manganese and/or galactose addition toCDM GIA-1 on galactosylation of adalimumab relative to control in CHOcell line #3.

FIG. 19A depicts the culture growth of adalimumab-producing NSO cellline in CDM PFBM-3/PFFM-4 fed-batch shake flasks. FIG. 19B depicts theviability of adalimumab-producing NSO cell line in CDM PFBM-3/PFFM-4fed-batch shake flasks. FIG. 19C depicts the normalized titer ofadalimumab-producing NSO cell line in CDM PFBM-3/PFFM-4 fed-batch shakeflasks.

FIG. 20A depicts the galactosylation profile (NGA2F+NGA2F-GlcNac) ofadalimumab in NSO cell line in CDM PFBM-3/PFFM-4 fed-batch shake flasks.FIG. 20B depicts the galactosylation profile (NA1F+NA2F) of adalimumabin NSO cell line in CDM PFBM-3/PFFM-4 fed-batch shake flasks.

FIG. 21 summarizes the effect of manganese and/or galactose addition toCDM PFBM-3/PFFM-4 on galactosylation of adalimumab relative to controlin NSO cell line.

FIG. 22A depicts the culture growth of CHO cell line producing mAb #1 inCDM GIA-1 in batch shake flasks. FIG. 22B depicts the viability of CHOcell line producing mAb #1 in CDM GIA-1 in batch shake flasks.

FIG. 23A depicts the galactosylation profile (NGA2F+NGA2F-GlcNac) of mAb#1 in CDM GIA-1 in batch shake flasks. FIG. 23B depicts thegalactosylation profile (NA1F+NA2F) of mAb #1 in CDM GIA-1 in batchshake flasks.

FIG. 24 summarizes the effect of manganese and/or galactose addition toCDM GIA-1 on galactosylation of mAb #1 relative to control.

FIG. 25A depicts the culture growth of CHO cell line producing mAb #2 inCDM GIA-1 in fed-batch 3 L bioreactors. FIG. 25B depicts the viabilityof CHO cell line producing mAb #2 in CDM GIA-1 in fed-batch 3 Lbioreactors. FIG. 25C depicts the normalized titer of CHO cell lineproducing mAb #2 in CDM GIA-1 in fed-batch 3 L bioreactors.

FIG. 26A depicts the glycosylation profile (NGA2F+NGA2F-GlcNAc) of mAb#2 in CDM GIA-1 in fed-batch 3 L bioreactors. FIG. 26B depicts theglycosylation profile (NA1F+NA2F) of mAb #2 in CDM GIA-1 in fed-batch 3L bioreactors.

FIG. 27 summarizes the effect of manganese and/or galactose addition toCDM GIA-1 on galactosylation of mAb #2 relative to control.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods modulating the glycosylationprofile of recombinantly-expressed proteins. In particular, the presentinvention relates to methods of controlling (e.g., modulating) thegalactosylation profile of recombinantly-expressed proteins bysupplementing production medium, e.g., a hydrolysate-based or a CDmedium, with manganese and/or galactose. For example, but not by way oflimitation, the present invention demonstrates that supplementation ofparticular ranges of manganese and/or galactose concentrations tochemically defined media can be used to fine-tune the galactosylationprofile of monoclonal antibodies produced in CHO and NSO cell lines.Similarly, supplementation of galactose alone to hydrolysate-based mediais effective to modulate the galactosylation profile of the monoclonalantibody adalimumab produced in a CHO cell line in a concentrationdependent manner. In view of such findings, the methods disclosed hereincan be used to modulate the galactose content of recombinant proteins bycontrolling the amounts of manganese and/or galactose present in cellculture media. The studies described herein have also established thatthe changes in the galactosylation profiles obtained via implementationof the methods of the present invention are not only scale (1.5 L vs.200 mL) and process independent (fed-batch in controlled bioreactorenvironment vs. batch in shake flasks), but also that no significantimpact on culture growth and productivity is observed for mostconditions studied.

A terminal galactose is added to NGA2F by β-galactosyltransferase enzymein the presence of manganese chloride, to produce NA1F (in the case ofan addition of a single terminal galactose) or NA2F (in the case of anaddition of two terminal galactose molecules). Thisgalactosyltransferase-mediated reaction employs UDP-galactose as thesugar substrate and Mn²⁺ as a cofactor for galactosyltransferase. Thus,without being bound by theory, it is believed that a change in proteinhomogeneity taking the form of an increase in the fraction of N-linkedoligosaccharide NGA2F and a decrease in the fraction of NA1F+NA2FN-linked oligosaccharides could be caused by either an insufficientamount of the substrate (UDP-galactose), the cofactor forgalactosyltransferase (Mn²⁺), or both.

In certain embodiments, the present invention is directed to methods ofcontrolling the galactosylation profile of recombinantly-expressedantibody. In certain embodiments, the recombinantly-expressed antibodyis an anti-TNFα antibody. In certain embodiments, therecombinantly-expressed anti-TNFα antibody is adalimumab.

In certain embodiments, the present invention is directed to methods ofcontrolling the galactosylation profile of recombinantly-expressedproteins by supplementing a production medium, e.g., a hydrolysate-basedor a CD medium, used in the production of recombinantly-expressedproteins with manganese and/or galactose. In certain embodiments, themanganese supplement can take the form of any biologically-acceptablemanganese salt, for example, but not limited to, manganese (II)chloride. In certain embodiments, the galactose supplement can take theform of any biologically-acceptable galactose-containing compound, forexample, but not limited to, D-(+)-galactose.

In certain embodiments, the present invention is directed to methods ofcontrolling the galactosylation profile of recombinantly-expressedproteins by supplementing a production medium, e.g., a hydrolysate-basedor a CD medium, used in the production of recombinantly-expressedproteins with a sufficient amount of manganese and/or amanganese-containing supplement to achieve at least about the followingmanganese concentrations in the production media: at least about 0.1, atleast about 0.2, at least about 0.5, at least about 1.0, at least about10, at least about 20, at least about 25, at least about 40, at leastabout 50, at least about 60, at least about 75, at least about 80, or atleast about 100 μM, wherein that production media is used to dilute asupplement-free cell culture growth media containing no supplement by aratio of about 1:4 or about 1:5 (supplement-free growthmedia:supplemented production media). In certain embodiments, thepresent invention is directed to methods of controlling thegalactosylation profile of recombinantly-expressed proteins bysupplementing a production medium, e.g., a hydrolysate-based or a CDmedium, used in the production of recombinantly-expressed proteins withsufficient galactose and/or galactose-containing supplement to achieveat least about the following galactose concentrations in the productionmedia: at least about 1, at least about 4, at least about 5, at leastabout 10, at least about 15, at least about 20, at least about 30, atleast about 40, at least about 60, or at least about 100 mM, whereinthat production media is used to dilute a supplement-free cell culturegrowth media containing no supplement by a ratio of about 1:4 or about1:5 (supplement-free growth media:supplemented production media).

In certain embodiments, the present invention is directed to methods ofcontrolling the galactosylation profile of recombinantly-expressedproteins by supplementing a production medium, e.g., a hydrolysate-basedor a CD medium, used in the production of recombinantly-expressedproteins with sufficient manganese and/or a manganese-containingsupplement and sufficient galactose and/or galactose-containingsupplement to achieve at least about the following manganese (Mn) andgalactose (Gal) concentrations in the production media presented as Mn(μM)/Gal (mM): 0/1, 0/4, 0/5, 0/10, 0/15, 0/20, 0/30, 0/40, 0/60, 0/100,0.1/0, 0.2/0, 0.5/0, 1.0/0, 10/0, 20/0, 25/0, 40/0, 50/0, 75/0, 80/0,100/0, 0.2/1, 0.2/4, 0.2/30, 0.5/1, 0.5/4, 0.5/30, 10/10, 10/20, 10/40,20/10, 20/20, 20/40, 25/15, 40/10, 40/20, 40/40, 40/100, 50/30, 60/20,60/40, 60/100, 80/20, 80/40, 80/100, 100/20, 100/40, 100/100, whereinthat production media is used to dilute a supplement-free cell culturegrowth media containing no supplement by a ratio of about 1:4 or about1:5 (supplement-free growth media:supplemented production media).

In certain embodiments, the present invention is directed to methods ofcontrolling the galactosylation profile of recombinantly-expressedproteins by supplementing a production medium, e.g., a hydrolysate-basedor a CD medium, used in the production of recombinantly-expressedproteins with sufficient manganese and/or a manganese-containingsupplement and sufficient galactose and/or galactose-containingsupplement to achieve at least about the following manganese (Mn) andgalactose (Gal) concentrations in the production media presented as Mn(μM)/Gal (mM): 0.2/1, 0.2/4, 0.2/30, 0.5/1, 0.5/4, 0.5/30, 10/10, 10/20,10/40, 20/10, 20/20, 20/40, 25/15, 40/10, 40/20, 40/40, 40/100, 50/30,60/20, 60/40, 60/100, 80/20, 80/40, 80/100, 100/20, 100/40, 100/100,wherein that production media is used to dilute a supplement-free cellculture growth media containing no supplement by a ratio of about 1:4 orabout 1:5 (supplement-free growth media:supplemented production media).

In certain embodiments, the production medium, e.g., a hydrolysate-basedor a CD medium, used in the production of a recombinantly-expressedprotein is supplemented with manganese and not galactose. For the shakeflasks studies in Example 1, but not by way of limitation, addition ofmanganese and not galactose to production IVGN CDM GIA-1 lowered theNGA2F+NGA2F-GlcNac sum by 6% to 9% and increased the NA1F+NA2F sum by 8%to 9% (FIGS. 3, 4, and 5). No further increase in manganeseconcentration was explored in the experimental design due to the growthinhibition observed at about 100 μM.

In certain embodiments, the production medium, e.g., a hydrolysate-basedor a CD medium, used in the production of a recombinantly-expressedprotein is supplemented with galactose and not manganese. For the shakeflasks studies in Example 1, but not by way of limitation, addition ofgalactose only to production IVGN CDM GIA-1 lowered theNGA2F+NGA2F-GlcNac sum by 3% to 7% and increased NA1F+NA2F by 3% to 7%(FIGS. 3, 4, and 5). These findings indicate that a manganeseconcentration of about 100 μM and a galactose concentration of about 100mM represent the maximum range of interest for this Example 1.

In certain embodiments, the production medium, e.g., a hydrolysate-basedmedia or a CD media, used in the production of a recombinantly-expressedprotein is supplemented with both manganese and galactose. For example,but not by way of limitation, the studies outlined in Example 1 indicatethat the addition of combinations of manganese and galactose toproduction IVGN CDM GIA-1 resulted in a significant decrease in theNGA2F+NGA2F-GlcNac sum of 11% to 26% and a corresponding significantincrease in the NA1F+NA2F sum of 13% to 23% as compared to the controlcondition where no manganese or galactose were added to the productionmedia (FIGS. 3, 4, and 5). The effect on modulation of galactosylationof adalimumab in production IVGN CDM GIA-1 with the combined addition ofmanganese chloride and galactose was synergistic. In particular, thecombined addition of manganese chloride and galactose decreased theNGA2F+NGA2F-GlcNac sum and increased the NA1F+NA2F sum by a largerpercentage than by adding manganese or galactose alone and summing uptheir individual effects. For example, but not by way of limitation,addition of 40 μM manganese chloride alone reduced the NGA2F sum by 6%,and addition of 40 mM galactose alone decreased the NGA2F+NGA2F-GlcNacsum by 6%. However, the combined addition of manganese chloride andgalactose at these same concentrations (i.e. 40 μM manganese+40 mMgalactose) led to an 18% reduction in the NGA2F+NGA2F-GlcNac sum, 6%higher than their combined individual contributions to the reduction ofthe NGA2F+NGA2F-GlcNac sum. We define this effect as being synergisticand maintain this definition throughout the invention. The largestpercent decrease in the NGA2F+NGA2F-GlcNac sum of approximately 26% wasobserved with the combined addition of 100 μM manganese chloride and 100mM galactose. The largest percent increase in the NA1F+NA2F sum ofapproximately 23% was recorded with the combined addition of 60 μMmanganese chloride and 100 mM galactose.

For the fed-batch bioreactor study described in Example 1, two manganesechloride and galactose combinations were studied and the resultsindicate that the decrease in the NGA2F+NGA2F-GlcNac sum and thecorresponding increase in the NA1F+NA2F sum was scale (1.5 L vs. 200 mL)and process independent (fed-batch in controlled bioreactor environmentvs. batch in shake flasks). For example, but not by way of limitation,the combined addition of 40 μM manganese chloride and 20 mM galactose toboth production basal CDM GIA-1 and feed CDM JCL-5 decreased theNGA2F+NGA2F-GlcNac sum by 26% and increased the NA2F+NA2F sum by 27%compared to the control cultures (FIG. 6). A further increase in thegalactose concentration to 40 mM in addition to manganesesupplementation at 40 μM concentration resulted in an additional 3%decrease in the NGA2F+NGA2F-GlcNac sum, and a corresponding 3% increasein the NA1F+NA2F sum

In certain embodiments, the present invention is directed to thesupplementation of CD media used in the production of arecombinantly-expressed protein with galactose and/or manganese. Thatsuch supplementation is effective across distinct CD media is evidencedby the results outlined in Example 2. Specifically, Example 2 resultsindicate that the addition of manganese chloride alone within the rangeof 0 to 40 μM to production CDM HyClone CDM4CHO decreased theNGA2F+NGA2F-GlcNac sum by a maximum of 5% in a concentration dependentmanner (FIG. 8). A comparable maximum increase of 4% in the NA1F+NA2Fsum was also achieved. Addition of galactose alone up to a maximumconcentration of 40 mM yielded a 6% maximum decrease in theNGA2F+NGA2F-GlcNac sum and a corresponding 6% increase in the NA1F+NA2Fsum. Modulation of galactosylation was also observed in production CDMHyClone CDM4CHO cultures supplemented with both manganese chloride andgalactose. An additive effect was observed in cultures supplemented withboth manganese chloride and galactose. The combined addition ofmanganese chloride and galactose decreased the NGA2F+NGA2F-GlcNac sumand increased the NA1F+NA2F sum by a comparable percentage as whenmanganese or galactose were added alone and their individual effectswere summed up (FIG. 9). For example, but not by way of limitation,addition of 40 μM manganese chloride alone reduced theNGA2F+NGA2F-GlcNac sum by 5%, and addition of 40 mM galactose alonedecreased the NGA2F sum by 6%. The combined addition of manganesechloride and galactose at these same concentrations (i.e. 40 μMmanganese+40 mM galactose) led to a 12% reduction in theNGA2F+NGA2F-GlcNac sum. We define this effect as being additive andmaintain this definition throughout the invention. The highestpercentage decrease in the NGA2F sum of 12% and the corresponding 11%increase in the NA1F+NA2F sum was observed for the culture supplementedwith 40 μM manganese chloride and 40 mM galactose.

In certain embodiments, the present invention is directed to thesupplementation of a hydrolysate-based media used in the production of arecombinantly-expressed protein with galactose and/or manganese. Forexample, as outlined in Example 3, the addition of manganese chloridealone within the range of 0 to 40 μM to hydrolysate-based productionmedia decreased the NGA2F+NGA2F-GlcNac sum by approximately 1%, althoughthat change is within the oligosaccharide assay variability (FIG. 11).The addition of galactose alone up to a maximum concentration of 40 mMyielded a maximum decrease of 4% in the NGA2F+NGA2F-GlcNac sum and acorresponding 4% maximum increase in the NA1F+NA2F sum. Sucholigosaccharide profile changes achieved with the addition of galactosealone are comparable to the changes recorded when combinations ofgalactose and manganese chloride were added to the hydrolysate-basedmedia. For example, the combined addition of manganese chloride rangingfrom 0 to 40 μM and galactose ranging from 0 to 40 mM tohydrolysate-based media led to an approximate 5% maximum decrease in theNGA2F+NGA2F-GlcNac sum and a corresponding 3% increase in the NA1F+NA2Fsum (FIG. 12). The highest percentage decrease of 5% in theNGA2F+NGA2F-GlcNac sum and the corresponding 4% increase in theNA1F+NA2F sum was observed for the culture supplemented with 40 mMgalactose and either 20 μM or 40 μM manganese chloride.

The compositions and methods of the present invention also find useacross distinct cell lines. For example, but not by way of limitation,the study described in Example 4 illustrates that the supplementation ofa CD media, GIA-1, with galactose and/or manganese is effective tomodulate galactosylation of adalimumab produced using a CHO cell linedistinct from that employed in Examples 1-3. For example, but not by wayof limitation, when using this alternative cell line, the addition ofmanganese chloride alone within the range of 0 to 20 μM to productionCDM GIA-1 decreased the NGA2F+NGA2F-GlcNac sum in a concentrationdependent manner and increased the NA1F+NA2F sum by approximately thesame percentage. A maximum decrease of 22% in the NGA2F+NGA2F-GlcNac sumand a maximum corresponding increase of 23% in the NA1F+NA2F sum wasobserved with the addition of 20 μM manganese chloride (FIG. 14).Similarly, a concentration dependent decrease in the NGA2F+NGA2F-GlcNacsum and a corresponding increase in the NA1F+NA2F sum was observed withthe addition of galactose alone in the range of 0 to 20 mM. A maximumdecrease of 9% in the NGA2F+NGA2F-GlcNac sum and a corresponding maximumincrease of 10% in the NA1F+NA2F sum was observed with the addition of20 mM galactose. Similarly, an additive effect was observed for theoligosaccharide profiles of adalimumab produced in cultures supplementedwith the combined addition of manganese chloride and galactose to GIA-1media (FIG. 15). For example, but not by way of limitataion, addition of10 μM manganese chloride alone reduced the NGA2F+NGA2F-GlcNac sum by18%, and addition of 10 mM galactose alone decreased theNGA2F+NGA2F-GlcNac sum by 6%. The combined addition of manganesechloride and galactose at these same concentrations led to a 24%reduction in the NGA2F+NGA2F-GlcNac sum. The highest percentage decreaseof 35% in the NGA2F+NGA2F-GlcNac sum and the corresponding increase of37% in the NA1F+NA2F sum were observed for the culture supplemented with40 μM manganese chloride and 20 mM galactose.

That the compositions and methods of the present invention also find useacross distinct cell lines is further reinforced by the results ofExample 5, which employs a third adalimumab-producing cell line that isdistinct from either of the adalimumab-producing cell lines of Examples1-4. For example, but not by way of limitation, when using this thirdcell line, the addition of manganese chloride alone within the range of0 to 1 μM to production CDM GIA-1 decreased the NGA2F+NGA2F-GlcNac sumin a concentration dependent manner and increased the NA1F+NA2F sum byapproximately the same percentage. A maximum decrease of 26% in theNGA2F+NGA2F-GlcNac and a corresponding increase of 28% in the NA1F+NA2Foligosaccharides were observed with the addition of 1 μM manganesechloride (FIG. 17). The addition of galactose alone at 30 mMconcentration to production CDM GIA-1 decreased the NGA2F+NGA2F-GlcNacsum by 4% and increased the NA1F+NA2F sum by 3%. Furthermore, whenmanganese chloride and galactose were supplemented together into theproduction basal and feed media, the results demonstrated a synergisticbenefit towards the decrease in the NGA2F+NGA2F-GlcNAc and the increasein the NA1F+NA2F oligosaccharides which is consistent with the resultsdemonstrated in Example 1 (FIG. 18). For example, but not by way oflimitation, at 0.2 μM manganese chloride plus 30 mM galactose theobserved 25% decrease in the NGA2F+NGA2F-GlcNAc sum was 6% more than thesum of the decrease observed with the addition of 0.2 μM manganesechloride alone (15%) and that of 30 mM galactose alone (4%). Similarly,the resulting 24% increase in the NA1F+NA2F sum was more than the sum ofthe increase observed with the addition of 0.2 μM manganese chloridealone (16%) and that of 30 mM galactose alone (3%). The combinedsupplementation of 0.5 μM manganese chloride+30 mM galactose alsodemonstrated a synergistic effect on the galactosylation profile ofadalimumab produced in this third cell line. A maximum decrease comparedto the control condition of 34% in the NGA2F+NGA2F-GlcNac and acorresponding 34% maximum increase in the NA1F+NA2F oligosaccharides wasobserved with the combined addition of 0.5 μM manganese chloride and 30mM galactose to chemically defined GIA-1 media.

That the compositions and methods of the present invention also find useacross distinct types of cell lines is further reinforced by the resultsof Example 6, which employs a fourth adalimumab-producing cell line thatis distinct from the adalimumab-producing cell lines of Examples 1-5, inthat it is an NSO cell line. For example, but not by way of limitation,when using this NSO cell line, the addition of manganese chloride alonewithin the range of 0 to 0.5 μM to production CDM PFBM-3/PFFM-4decreased the NGA2F+NGA2F-GlcNac sum in a concentration dependent mannerand increased the NA1F+NA2F sum by approximately the same percentage. Amaximum decrease of 18% in the NGA2F+NGA2F-GlcNac sum and acorresponding increase of 20% in the

NA1F+NA2F sum were observed with the addition of 0.5 μM manganesechloride (FIG. 20). However, manganese doses greater than 0.5 μM werenot explored further due to cytotoxicity effects. Similarly, aconcentration dependent decrease in the NGA2F+NGA2F-GlcNac sum and acorresponding increase in the NA1F+NA2F sum were observed with theaddition of galactose alone in the range of 0 to 10 mM to production CDMPFBM-3/PFFM-4. A maximum decrease of 14% in the NGA2F+NGA2F-GlcNac sumand a corresponding increase of 15% in the NA1F+NA2F sum was observedwith the addition of 10 mM galactose. In addition, the effect onmodulation of galactosylation of adalimumab produced in a NSO cell linein production CDM PFBM-3/PFFM-4 supplemented with manganese chloride andgalactose was synergistic (FIG. 21). For example, but not by way oflimitation, addition of 0.2 μM manganese chloride alone reduced theNGA2F+NGA2F-GlcNac sum by 12%, and addition of 4 mM galactose alonedecreased the NGA2F+NGA2F-GlcNac sum by 2%. However, the combinedaddition of manganese chloride and galactose at these sameconcentrations (i.e. 0.2 μM manganese+4 mM galactose) led to a 19%reduction in the NGA2F+NGA2F-GlcNac sum, 5% higher than their combinedindividual contributions. A maximum decrease of ˜26% in theNGA2F+NGA2F-GlcNac sum and a corresponding 28% increase in the NA1F+NA2Fsum were observed with the combined addition of 0.5 μM manganesechloride and 4 mM galactose.

The compositions and methods of the present invention also find use inthe production of diverse antibodies, as evidenced by the results ofExample 7, which employs a CHO cell line that produced an antibodydistinct from adalimumab. For example, but not by way of limitation,when producing this antibody distinct from adalimumab, the addition ofmanganese chloride alone within the range of 0 to 40 μM to productionCDM GIA-1 decreased the NGA2F+NGA2F-GlcNac sum by a maximum of 26% (FIG.23). A comparable maximum increase of 27% in the NA1F+NA2F sum was alsoachieved. Addition of galactose alone up to a maximum concentration of40 mM yielded a maximum decrease of 12% in the NGA2F+NGA2F-GlcNac sumand a corresponding 13% maximum increase in the NA1F+NA2F sum in aconcentration dependent manner. In addition, the combined addition ofgalactose and manganese chloride to production CDM GIA-1 resulted in agreater percent reduction in the NGA2F+NGA2F-GlcNAc sum and,correspondingly, a greater percent increase in the NA1F+NA2F sum ascompared to the addition of either component alone (FIG. 24). Forexample, but not by way of limitation, the addition of 40 μM manganesechloride alone reduced the NGA2F+NGA2F-GlcNAc sum by 20%, and theaddition of 40 mM galactose alone decreased the NGA2F+NGA2F-GlcNac sumby 12%. However, the combined addition of manganese chloride andgalactose at these same concentrations (i.e. 40 μM manganese+40 mMgalactose) led to a 27% decrease in the NGA2F+NGA2F-GlcNac sum. Thehighest percentage decrease of 32% in the NGA2F+NGA2F-GlcNac sum and thecorresponding increase of 30% in the NA1F+NA2F sum were observed for theculture supplemented with 20 μM manganese chloride and 20 mM galactose.

That the compositions and methods of the present invention also find usewhen producing diverse antibodies is further reinforced by the resultsof Example 8, which employs a CHO cell line producing an antibodydistinct from both adalimumab and the antibody of Example 7. Forexample, but not by way of limitation, when producing this thirdantibody, the addition of manganese chloride alone in the range of 0 to75 μM to production CDM GIA-1 decreased the NGA2F+NGA2F-GlcNac sum by amaximum of 18% (FIG. 26). A comparable maximum increase of 16% in theNA1F+NA2F sum was also achieved. Addition of galactose alone up to amaximum concentration of 60 mM yielded a maximum decrease of 12% in theNGA2F+NGA2F-GlcNac sum and a corresponding 11% maximum increase in theNA1F+NA2F sum. In addition, when manganese chloride and galactose weresupplemented together into the basal and feed media, the resultsdemonstrated at least an additive effect and sometimes a synergisticeffect towards the decrease in the NGA2F+NGA2F-GlcNAc sum and theincrease in the NA1F+NA2F sum (FIG. 27). The synergistic effect wasobserved for the condition supplemented with 25 μM manganese chlorideand 15 mM galactose. The observed 22% decrease in the NGA2F+NGA2F-GlcNAcsum was 5% more than the sum of the decrease observed with the additionof 25 μM manganese chloride alone (10%) and 15 mM galactose alone (7%).The additive effect was observed for the condition supplemented with 50μM manganese chloride and 30 mM galactose. The observed 28% decrease inthe NGA2F+NGA2F-GlcNAc sum was comparable to the sum of the decreaseobserved with the addition of 50 μM manganese chloride alone (18%) and30 mM galactose alone (12%). A maximum decrease of 28% in theNGA2F+NGA2F-GlcNac and a corresponding 25% maximum increase in theNA1F+NA2F sum compared to the control condition was observed with thecombined addition of 50 μM manganese chloride and 30 mM galactose tochemically defined GIA-1 media.

Although specifically directed to the production of antibodies, thefollowing description outlines general techniques that can be adaptedfor the production of other recombinantly-expressed proteins. Forexample, to express a recombinant antibody, nucleic acids encodingpartial or full-length light and heavy chains are inserted into one ormore expression vector such that the genes are operatively linked totranscriptional and translational control sequences. (See, e.g., U.S.Pat. No. 6,914,128, the entire teaching of which is incorporated hereinby reference.) In this context, the term “operatively linked” isintended to mean that an antibody gene is ligated into a vector suchthat transcriptional and translational control sequences within thevector serve their intended function of regulating the transcription andtranslation of the antibody gene. The expression vector and expressioncontrol sequences are chosen to be compatible with the expression hostcell used. The antibody light chain gene and the antibody heavy chaingene can be inserted into a separate vector or, more typically, bothgenes are inserted into the same expression vector. The antibody genesare inserted into an expression vector by standard methods (e.g.,ligation of complementary restriction sites on the antibody genefragment and vector, or blunt end ligation if no restriction sites arepresent). Prior to insertion of the antibody or antibody-related lightor heavy chain sequences, the expression vector may already carryantibody constant region sequences. For example, one approach toconverting particular VH and VL sequences to full-length antibody genesis to insert them into expression vectors already encoding heavy chainconstant and light chain constant regions, respectively, such that theVH segment is operatively linked to the CH segment(s) within the vectorand the VL segment is operatively linked to the CL segment within thevector. Additionally or alternatively, the recombinant expression vectorcan encode a signal peptide that facilitates secretion of the antibodychain from a host cell. The antibody chain gene can be cloned into thevector such that the signal peptide is linked in-frame to the aminoterminus of the antibody chain gene. The signal peptide can be animmunoglobulin signal peptide or a heterologous signal peptide (i.e., asignal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, a recombinant expression vectorof the invention can carry one or more regulatory sequence that controlsthe expression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, e.g., in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990), the entire teaching of which is incorporatedherein by reference. It will be appreciated by those skilled in the artthat the design of the expression vector, including the selection ofregulatory sequences may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Suitable regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., theadenovirus major late promoter (AdMLP)) and polyoma. For furtherdescription of viral regulatory elements, and sequences thereof, see,e.g. U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 by Bellet al. and U.S. Pat. No. 4,968,615 by Schaffner et al., the entireteachings of which are incorporated herein by reference.

In addition to the antibody chain genes and regulatory sequences, arecombinant expression vector of the invention may carry one or moreadditional sequences, such as a sequence that regulates replication ofthe vector in host cells (e.g., origins of replication) and/or aselectable marker gene. The selectable marker gene facilitates selectionof host cells into which the vector has been introduced (see e.g., U.S.Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al., theentire teachings of which are incorporated herein by reference). Forexample, typically the selectable marker gene confers resistance todrugs, such as G418, hygromycin or methotrexate, on a host cell intowhich the vector has been introduced. Suitable selectable marker genesinclude the dihydrofolate reductase (DHFR) gene (for use in dhfr− hostcells with methotrexate selection/amplification) and the neo gene (forG418 selection).

An antibody, or antibody portion, of the invention can be prepared byrecombinant expression of immunoglobulin light and heavy chain genes ina host cell. To express an antibody recombinantly, a host cell istransfected with one or more recombinant expression vectors carrying DNAfragments encoding the immunoglobulin light and heavy chains of theantibody such that the light and heavy chains are expressed in the hostcell and secreted into the medium in which the host cells are cultured,from which medium the antibodies can be recovered. Standard recombinantDNA methodologies are used to obtain antibody heavy and light chaingenes, incorporate these genes into recombinant expression vectors andintroduce the vectors into host cells, such as those described inSambrook, Fritsch and Maniatis (eds), Molecular Cloning; A LaboratoryManual, Second Edition, Cold Spring Harbor, N.Y., (1989), Ausubel et al.(eds.) Current Protocols in Molecular Biology, Greene PublishingAssociates, (1989) and in U.S. Pat. Nos. 4,816,397 & 6,914,128, theentire teachings of which are incorporated herein.

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is (are) transfected into a hostcell by standard techniques. The various forms of the term“transfection” are intended to encompass a wide variety of techniquescommonly used for the introduction of exogenous DNA into a prokaryoticor eukaryotic host cell, e.g., electroporation, calcium-phosphateprecipitation, DEAE-dextran transfection and the like. Although it istheoretically possible to express the antibodies of the invention ineither prokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells, such as mammalian host cells, is suitable because sucheukaryotic cells, and in particular mammalian cells, are more likelythan prokaryotic cells to assemble and secrete a properly folded andimmunologically active antibody. Prokaryotic expression of antibodygenes has been reported to be ineffective for production of high yieldsof active antibody (Boss and Wood (1985) Immunology Today 6:12-13, theentire teaching of which is incorporated herein by reference).

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, e.g., Enterobacteriaceae suchas Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella,Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g.,Serratia marcescans, and Shigella, as well as Bacilli such as B.subtilis and B. licheniformis (e.g., B. licheniformis 41P disclosed inDD 266,710 published Apr. 12, 1989), Pseudomonas such as P. aeruginosa,and Streptomyces. One suitable E. coli cloning host is E. coli 294 (ATCC31,446), although other strains such as E. coli B, E. coli X1776 (ATCC31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examplesare illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for polypeptideencoding vectors. Saccharomyces cerevisiae, or common baker's yeast, isthe most commonly used among lower eukaryotic host microorganisms.However, a number of other genera, species, and strains are commonlyavailable and useful herein, such as Schizosaccharomyces pombe;Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424),K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii(ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K.marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida;Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces suchas Schwanniomyces occidentalis; and filamentous fungi such as, e.g.,Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A.nidulans and A. niger.

Suitable host cells for the expression of glycosylated antibodies arederived from multicellular organisms. Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyxmori have been identified. A variety of viral strains for transfectionare publicly available, e.g., the L-1 variant of Autographa californicaNPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be usedas the virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells. Plant cell cultures ofcotton, corn, potato, soybean, petunia, tomato, and tobacco can also beutilized as hosts.

Suitable mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr− CHO cells, described in Urlaub and Chasin, (1980) PNAS USA77:4216-4220, used with a DHFR selectable marker, e.g., as described inKaufman and Sharp (1982) Mol. Biol. 159:601-621, the entire teachings ofwhich are incorporated herein by reference), NS0 myeloma cells, COScells and SP2 cells. When recombinant expression vectors encodingantibody genes are introduced into mammalian host cells, the antibodiesare produced by culturing the host cells for a period of time sufficientto allow for expression of the antibody in the host cells or secretionof the antibody into the culture medium in which the host cells aregrown. Other examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/−DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2), the entire teachings of which are incorporated herein byreference.

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

The host cells used to produce an antibody may be cultured in a varietyof media. Commercially available media such as Ham's F10™ (Sigma),Minimal Essential Medium™ ((MEM), (Sigma), RPMI-1640 (Sigma), andDulbecco's Modified Eagle's Medium™ ((DMEM), Sigma) are suitable forculturing the host cells. In addition, any of the media described in Hamet al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255(1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. No. Re. 30,985 may beused as culture media for the host cells, the entire teachings of whichare incorporated herein by reference. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

Host cells can also be used to produce portions of intact antibodies,such as Fab fragments or scFv molecules. It is understood thatvariations on the above procedure are within the scope of the presentinvention. For example, in certain embodiments it may be desirable totransfect a host cell with DNA encoding either the light chain or theheavy chain (but not both) of an antibody of this invention. RecombinantDNA technology may also be used to remove some or all of the DNAencoding either or both of the light and heavy chains that is notnecessary for antigen binding. The molecules expressed from suchtruncated DNA molecules are also encompassed by the antibodies of theinvention. In addition, bifunctional antibodies may be produced in whichone heavy and one light chain are an antibody of the invention and theother heavy and light chain are specific for an antigen other than theoriginal antigen by crosslinking an antibody of the invention to asecond antibody by standard chemical crosslinking methods.

In a suitable system for recombinant expression of an antibody, orantigen-binding portion thereof, of the invention, a recombinantexpression vector encoding both the antibody heavy chain and theantibody light chain is introduced into dhfr-CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the antibody heavy and light chain genes are each operativelylinked to CMV enhancer/AdMLP promoter regulatory elements to drive highlevels of transcription of the genes. The recombinant expression vectoralso carries a DHFR gene, which allows for selection of CHO cells thathave been transfected with the vector using methotrexateselection/amplification. The selected transformant host cells arecultured to allow for expression of the antibody heavy and light chainsand intact antibody is recovered from the culture medium. Standardmolecular biology techniques are used to prepare the recombinantexpression vector, transfect the host cells, select for transformants,culture the host cells and recover the antibody from the culture medium.

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. In one aspect, if the antibody is produced intracellularly, as afirst step, the particulate debris, either host cells or lysed cells(e.g., resulting from homogenization), can be removed, e.g., bycentrifugation or ultrafiltration. Where the antibody is secreted intothe medium, supernatants from such expression systems can be firstconcentrated using a commercially available protein concentrationfilter, e.g., an Amicon™ or Millipore Pellicon™ ultrafiltration unit.

Prior to the process of the invention, procedures for purification ofantibodies from cell debris initially depend on the site of expressionof the antibody. Some antibodies can be secreted directly from the cellinto the surrounding growth media; others are made intracellularly. Forthe latter antibodies, the first step of a purification processtypically involves: lysis of the cell, which can be done by a variety ofmethods, including mechanical shear, osmotic shock, or enzymatictreatments. Such disruption releases the entire contents of the cellinto the homogenate, and in addition produces subcellular fragments thatare difficult to remove due to their small size. These are generallyremoved by differential centrifugation or by filtration. Where theantibody is secreted, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, e.g., an Amicon™ or Millipore Pellicon™ultrafiltration unit. Where the antibody is secreted into the medium,the recombinant host cells can also be separated from the cell culturemedium, e.g., by tangential flow filtration. Antibodies can be furtherrecovered from the culture medium using the antibody purificationmethods of the invention.

6. EXAMPLES 6.1. Example 1

6.1.1. Materials & Methods

In the studies summarized in this example, we investigated the effectson product quality attributes resulting from the addition of manganesechloride and/or galactose to chemically defined Life Technologies Gibco,GIA-1, media (proprietary formulation) in the adalimumab-producing CHOcell line utilized in Example 3, but adapted to GIA-1 media. The studieswere performed in either a batch process in shake flasks or a fed-batchprocess in 3 L bioreactors.

Growth and production media for the adalimumab-producing CHO cell linewere prepared using a proprietary Life Technologies Gibco chemicallydefined media, GIA-1. Basal production and feed media were supplementedwith Manganese (II) Chloride (Sigma M1787-100 mL; 1.0 M±0.1 M) andD(+)Galactose (Sigma G5388-1 kg) according to the experimental designdescribed in Table 1. All media were filtered through Corning 0.5 L or 1L filter systems 0.22 μm Poly(Ether Sulfone) (PES) and stored at 4° C.until use.

The cell line utilized for both studies was generated from theadalimumab-producing CHO cell utilized in Example 3 by adapting it tochemically defined GIA-1 media for 7 (2 to 3 day each) passages in acombination of 250 mL and 500 mL Corning vented non-baffled shake flasksbefore freezing.

Upon thaw, for the batch shake flask study, cells were expanded for 3 to5 (2 to 3 day each) passages in a combination of 250 mL and 500 mLCorning vented non-baffled shake flasks. Production cultures wereinitiated in duplicate 500 mL Corning vented non-baffled shake flasks(200 mL working volume) at an initial viable cell density (VCD) ofapproximately 0.5×106 cells/mL. Cultures were maintained on orbitalshakers at 110 revolutions per minute (RPM) in a dry incubator at 35° C.and 5% CO₂. The shake flask study was run in an extended batch mode byfeeding a glucose solution (1.25% (v/v) of 40% solution) when the mediaglucose concentration fell below 3 g/L.

For the fed-batch bioreactor study, cells were expanded for 8 (2 to 3day each) passages in Corning vented non-baffled shake flasks maintainedon orbital shakers at 110 RPM and in 20 L cell bags (3 L to 10 L workingvolume) maintained at 20-25 RPM, 7.5° angle, and 0.25 SLPM airflow in adry incubator at 35° C. and 5% CO₂. Production cultures were initiatedin duplicate 3 L bioreactors (1.5 L working volume) at 35° C., 30%dissolved oxygen, 200 RPM, pH ramp from 7.1 to 6.9 over 3 days, and pHsetpoint of 6.9 thereafter. A fixed split ratio of cells to media of 1:5was utilized to initiate the production stage cultures. In the fed-batchmode, a chemically-defined feed from Life Technologies Gibco, JCL-5(proprietary formulation), was added as follows: 3% (v/v)—day 3, 5%—day4, 7%—day 5, 10%—day 6, and 10%—day 7. Additional glucose (1.25% (v/v)of 40% solution) was fed when the media glucose concentration fell below3 g/L.

For all studies with CHO cell lines described throughout this invention,samples were collected daily and measured for cell density and viabilityusing a Cedex cell counter. Retention samples for titer analysis viaPoros A method were collected by centrifugation at 12,000 RPM for 5 minwhen the culture viability began declining. The cultures were harvestedby collecting 125 mL aliquots and centrifuging at 3,000 RPM for 30 minwhen culture viability was near or below 50%. All supernatants werestored at −80° C. until analysis.

For all studies, the harvest samples were Protein A purified andprepared for the oligosaccharide assay using the following procedures.As a first step in the process of establishing the identity andquantifying the oligosaccharides, they are released from the protein byenzymatic digestion with N-glycanase. Once the glycans are released, thefree reducing end of each glycan is labeled by reductive amination witha fluorescent tag, 2-aminobenzamide (2-AB). The resulting labeledglycans are separated by normal-phase HPLC (NP-HPLC) in acetonitrile: 50mM ammonium formate, pH 4.4, and detected by a fluorescence detector.Quantitation is based on the relative area percent of detected sugars.Throughout this invention, the relative area percentages of theagalactosyl fucosylated biantennary oligosaccharides, denoted asNGA2F+NGA2F-GlcNAc sum, and the galactose-containing fucosylatedbiantennary oligosaccharides NA1F+NA2F sum are reported and discussed.

6.1.2. Experimental Design

As detailed in Table 1, for the batch shake flask study, manganesechloride was supplemented at the following concentrations in productionmedia: 0, 40, 60, 80, and 100 μM. Galactose was supplemented at thefollowing levels in production media: 0, 10, 20, 40, and 100 mM.Individual and combined additions of manganese chloride and galactosewere studied using a comprehensive design divided into 3 sets ofexperiments. Each experiment had a control culture for direct comparisonof culture growth, productivity, and product quality. Production mediaused for the control cultures was not supplemented with manganesechloride or galactose. Culture growth, productivity, and product qualitydata for control cultures is the average of the 3 experiments.

For the fed-batch bioreactor study, manganese chloride and galactosewere supplemented to both production and feed media in the followingcombinations: 40 μM manganese chloride and 20 mM galactose; 40 μMmanganese chloride and 40 mM galactose (Table 2). Basal and feed mediafor the control cultures were not supplemented with manganese chlorideor galactose.

TABLE 1 Experimental design for the batch shake flasks study (Example 1)Manganese Galactose (μM) (mM) ID 0 0 Mn(0) Gal(0) 0 10 Mn(0) Gal(10) 020 Mn(0) Gal(20) 0 40 Mn(0) Gal(40) 0 100 Mn(0) Gal(100) 40 0 Mn(40) Gal(0) 80 0 Mn(80) Gal(0) 100 0 Mn(100) Gal(0) 40 10 Mn(40) Gal(10) 40 20Mn(40) Gal(20) 40 40 Mn(40) Gal(40) 40 100 Mn(40) Gal(100) 60 20 Mn(60)Gal(20) 60 40 Mn(60) Gal(40) 60 100 Mn(60) Gal(100) 80 20 Mn(80) Gal(20)80 40 Mn(80) Gal(40) 80 100 Mn(80) Gal(100) 100 20 Mn(100) Gal(20) 10040 Mn(100) Gal(40) 100 100 Mn(100) Gal(100)

TABLE 1 Experimental design for the fed-batch 3 L bioreactors study(Example 1) Manganese Galactose (μM) (mM) ID 0 0 Mn(0) Gal(0) 40 20Mn(40) Gal(20) 40 40 Mn(40) Gal(40)

6.1.3. Culture Growth & Productivity

For the shake flasks experiments, cell growth and viability profiles ofcultures in production media supplemented with galactose in the 0 to 100mM concentration range and/or manganese chloride up to 80 μMconcentration were comparable to control cultures without manganeseand/or galactose added (FIGS. 1A, 1B). Cultures grown in mediasupplemented with manganese chloride at 100 μM concentration andgalactose concentrations in the 0 to 100 mM range experienced growth lagand decreased viability for the first 4 production days, likely due totoxic effects of manganese at this concentration. However, toxicityeffects were overcome after production day 4. Harvest titer for mostexperimental conditions was 3% to 14% higher than the average harvesttiter for the control cultures (FIG. 1C). All three control cultures hadcomparable growth profiles and productivity. For the bioreactorsfed-batch experiment, culture growth, viability profiles, and harvesttiter were comparable for all conditions (FIGS. 2A, 2B, 2C).

6.1.4. Oligosaccharide Analysis

In this example, the modulation of galactosylation with the addition ofmanganese chloride and/or galactose to chemically defined media GIA-1was explored using the adalimumab-producing CHO cell line utilized inExample 3, but adapted to GIA-1 media.

6.1.4.1. Batch Shake Flask Study

For the shake flask study, the addition of manganese chloride alonewithin the range of 0 to 100 μM to production CDM GIA-1 decreased theNGA2F+NGA2F-GlcNac sum in a concentration dependent manner and increasedthe NA1F+NA2F sum by approximately the same percentage. A maximum changeof 9% in the NGA2F+NGA2F-GlcNac and 8% in the NA1F+NA2F oligosaccharideswas observed with the addition of 100 μM manganese chloride (FIG. 3).Manganese doses greater than 100 μM were not explored further due tocytotoxicity effects. Similarly, a concentration dependent decrease inthe NGA2F+NGA2F-GlcNac sum and a corresponding increase in the NA1F+NA2Fsum were observed with the addition of galactose alone in the range of 0to 100 mM to production CDM GIA-1. A maximum change of 7% in theNGA2F+NGA2F-GlcNac and the NA1F+NA2F oligosaccharides was observed withthe addition of 100 mM galactose.

The effect on modulation of galactosylation of adalimumab in CDM GIA-1with the combined addition of manganese chloride and galactose wassynergistic. Combined addition of manganese chloride and galactosedecreased the NGA2F+NGA2F-GlcNac sum and increased the NA1F+NA2F sum bya larger percentage than by adding manganese or galactose alone andsumming up their individual effects (FIGS. 4 and 5). For example,addition of 40 μM manganese chloride alone reduced theNGA2F+NGA2F-GlcNac sum by 6%, and addition of 40 mM galactose alonedecreased the NGA2F+NGA2F-GlcNac sum by 6%. However, the combinedaddition of manganese chloride and galactose at these sameconcentrations (i.e. 40 μM manganese+40 mM galactose) led to an 18%reduction in the NGA2F+NGA2F-GlcNac sum, 6% higher than their combinedindividual contributions to the reduction of the NGA2F+NGA2F-GlcNac sum.We define this effect as being synergistic and maintain this definitionthroughout the invention. A maximum decrease compared to the controlcondition of 26% in the NGA2F+NGA2F-GlcNac sum was observed with thecombined addition of 100 μM manganese chloride and 100 mM galactose.

6.1.4.2. Fed-Batch Bioreactor Study

For the fed-batch 3 L bioreactors study, two manganese chloride andgalactose combinations were studied and we show that the decrease in theNGA2F+NGA2F-GlcNac and the corresponding increase in the NA1F+NA2Foligosaccharides was scale (1.5 L vs. 200 mL) and process independent(fed-batch in controlled bioreactor environment vs. batch in shakeflasks). Combined addition of 40 μM manganese chloride and 20 mMgalactose to both production basal CDM GIA-1 and feed CDM JCL-5decreased the NGA2F+NGA2F-GlcNac sum by 26% and increased the NA2F+NA2Fsum by 27% compared to the control cultures (FIG. 6). A further increasein the galactose concentration to 40 mM in addition to manganesesupplementation at 40 μM concentration resulted in an additional 3%decrease in the NGA2F+NGA2F-GlcNac sum, and a corresponding 3% increasein the NA1F+NA2F sum.

6.2. Example 2

6.2.1. Materials & Methods

In the study summarized in this example, we investigated the effects onproduct quality attributes resulting from the addition of manganesechloride and/or galactose to the chemically defined media ThermoScientific HyClone CDM4CHO using the adalimumab-producing CHO cell lineused in Example 1 further adapted to HyClone media.

Growth and production media for the adalimumab-producing CHO cell linewere prepared using Thermo Scientific HyClone chemically defined mediaCDM4CHO without L-glutamine (Catalogue #SH30558.02). Production mediawas supplemented with Manganese (II) Chloride (Sigma M1787-100 mL; 1.0M±0.1 M) and D(+)Galactose (Sigma G5388-1 kg) according to theexperimental design described in Table 3. All media were filteredthrough Corning 0.5 L or 1 L (0.22 μm PES) filter systems and stored at4° C. until use.

Upon thaw, cells were adapted to and expanded in HyClone CDM4CHO mediafor 5 (2 to 3 day each) passages in a combination of 250 mL, 500 mL, and1000 mL Corning vented non-baffled shake flasks. Production cultureswere initiated in duplicate 500 mL Corning vented non-baffled shakeflasks (200 mL working volume) at an initial VCD of approximately0.5×106 cells/mL. Cultures were maintained on orbital shakers at 110 RPMin a dry incubator at 35° C. and 5% CO₂. A glucose solution (1.25% (v/v)of 40% solution) was fed when the media glucose concentration fell below3 g/L.

6.2.2. Experimental Design

As detailed in Table 3, manganese chloride was supplemented at thefollowing concentrations in production media: 0, 10, 20, and 40 μM.Galactose was supplemented at the following levels in production media:0, 10, 20, and 100 mM. Production media for the control cultures was notsupplemented with manganese chloride or galactose.

TABLE 3 Experimental design for Example 2 Manganese Galactose (μM) (mM)ID 0 0 Mn(0) Gal(0) 0 10 Mn(0) Gal(10) 0 20 Mn(0) Gal(20) 0 40 Mn(0)Gal(40) 10 0 Mn(10) Gal (0) 20 0 Mn(20) Gal(0) 40 0 Mn(40) Gal(0) 10 10Mn(10) Gal(10) 20 20 Mn(20) Gal(20) 40 40 Mn(40) Gal(40)

6.2.3. Culture Growth & Productivity

Cell growth and viability profiles of cultures in production HyCloneCDM4CHO supplemented with galactose in the 0 to 40 mM concentrationrange and/or manganese chloride up to 10 μM concentration werecomparable to the control cultures without manganese and/or galactoseadded (FIGS. 7A, 7B). Increasing the concentration of manganese chloridein HyClone CDM4CHO production media to 20 μM or 40 μM slowed downculture growth. Manganese doses greater than 40 μM were not exploredfurther due to the observed growth inhibition effects. Harvest titer forall conditions was comparable to the control (data not shown).

6.2.4. Oligosaccharide Analysis

In this example, the modulation of galactosylation with the addition ofmanganese chloride and/or galactose to the commercially availableHyClone CDM4CHO was explored using the adalimumab-producing CHO cellline used Example 1 further adapted to HyClone media.

The addition of manganese chloride alone within the range of 0 to 40 μMto production CDM HyClone CDM4CHO decreased the NGA2F+NGA2F-GlcNac sumby a maximum of 5% in a concentration dependent manner (FIG. 8). Acomparable maximum increase of 4% in the NA1F+NA2F sum was achieved.Addition of galactose alone up to a maximum concentration of 40 mMyielded a 6% maximum decrease in the NGA2F+NGA2F-GlcNac sum and acorresponding 6% increase in the NA1F+NA2F sum.

An additive effect was observed in cultures supplemented with bothmanganese chloride and galactose. The combined addition of manganesechloride and galactose decreased the NGA2F+NGA2F-GlcNac sum andincreased the NA1F+NA2F sum by a comparable percentage as when manganeseor galactose were added alone and their individual effects were summedup (FIG. 9). For example, addition of 40 μM manganese chloride alonereduced the NGA2F+NGA2F-GlcNac sum by 5%, and addition of 40 mMgalactose alone decreased the NGA2F+NGA2F-GlcNac sum by 6%. The combinedaddition of manganese chloride and galactose at these sameconcentrations (i.e. 40 μM manganese+40 mM galactose) led to a 12%reduction in the NGA2F+NGA2F-GlcNac sum. We define this effect as beingadditive and maintain this definition throughout the invention.

The highest percentage decrease in the NGA2F+NGA2F-GlcNac sum of 12% andthe corresponding 11% increase in the NA1F+NA2F sum was observed for theculture supplemented with 40 μM manganese chloride and 40 mM galactose.

6.3. Example 3

6.3.1. Materials & Methods

In the study summarized in this example, we investigated the effects onproduct quality attributes resulting from the addition of manganesechloride and/or galactose to a hydrolysate-based media (proprietaryformulation) in an adalimumab-producing CHO cell line.

Growth and production media for the adalimumab-producing CHO cell linewere prepared using yeast and soy hydrolysates according to aproprietary formulation. Production media was supplemented withManganese (II) Chloride (Sigma M1787-100 mL; 1.0 M±0.1 M) andD(+)Galactose (Sigma G5388-1 kg) according to the experimental designdescribed in Table 4. All media were filtered through Corning 0.5 L or 1L filter systems (0.22 μm PES) and stored at 4° C. until use.

Upon thaw, cells were expanded for 9 (2 to 3 day each) passages in acombination of 250 mL, 500 mL, and 1000 mL Corning vented non-baffledshake flasks. Production cultures were initiated in duplicate 500 mLCorning vented non-baffled shake flasks (200 mL working volume) at aninitial VCD of approximately 0.5×106 cells/mL. Cultures were maintainedon orbital shakers at 110 RPM in a dry incubator at 35° C. and 5% CO₂. Aglucose solution (1.25% (v/v) of 40% solution) was fed when the mediaglucose concentration fell below 3 g/L.

6.3.2. Experimental Design

As detailed in Table 4, manganese chloride was supplemented at thefollowing concentrations in production media: 0, 10, 20, and 40 μM.Galactose was supplemented at the following levels in production media:0, 10, 20, and 40 mM. Production media for the control cultures was notsupplemented with manganese chloride or galactose.

TABLE 4 Experimental design for Example 3 Manganese Galactose (μM) (mM)ID 0 0 Mn(0) Gal(0) 0 10 Mn(0) Gal(10) 0 20 Mn(0) Gal(20) 0 40 Mn(0)Gal(40) 10 0 Mn(10) Gal (0) 20 0 Mn(20) Gal(0) 40 0 Mn(40) Gal(0) 10 10Mn(10) Gal(10) 20 20 Mn(20) Gal(20) 40 40 Mn(40) Gal(40)

6.3.3. Culture Growth & Productivity

Cell growth of most cultures in the hydrolysate-based media supplementedwith galactose in the 0 to 40 mM concentration range and/or manganesechloride in the 0 to 40 μM concentration was slower compared to thecontrol cultures without manganese or galactose added (FIG. 10A).However, all cultures reached a comparable peak viable cell density.Some cultures supplemented with 20 μM or 40 μM manganese chloride showeddecreased viability by day 3 of production culture, but recovered as thecultures progressed (FIG. 10B). The culture supplemented with 10 μMmanganese chloride was studied with a second control condition (B) in aseparate experiment. Both these cultures grew to slightly higher maximumVCD compared to all other cultures, however results were withinhistorical variation. Harvest titer for all conditions was comparable tothe control (data not shown).

6.3.4. Oligosaccharide Analysis

In this example, the modulation of galactosylation by the addition ofmanganese chloride and/or galactose to a hydrolysate-based media wasexplored using an adalimumab-producing CHO cell line in shake flasks.

The addition of manganese chloride alone within the range of 0 to 40 μMto hydrolysate-based production media decreased the NGA2F+NGA2F-GlcNacsum by approximately 1%, a change that is within the oligosaccharideassay variability (FIG. 11). The addition of galactose alone up to amaximum concentration of 40 mM yielded a maximum decrease of 4% in theNGA2F+NGA2F-GlcNac sum and a corresponding 4% maximum increase in theNA1F+NA2F sum.

The oligosaccharide profile changes achieved with the addition ofgalactose alone are comparable to the changes recorded when combinationsof galactose and manganese chloride were added to the hydrolysate-basedmedia. The combined addition of manganese chloride ranging from 0 to 40μM and galactose ranging from 0 to 40 mM to hydrolysate-based media ledto an approximate 5% maximum decrease in the NGA2F+NGA2F-GlcNac sum anda corresponding 3% increase in the NA1F+NA2F sum (FIG. 12). The highestpercentage decrease of 5% in the NGA2F+NGA2F-GlcNac sum and thecorresponding 4% increase in the NA1F+NA2F sum was observed for theculture supplemented with 40 mM galactose and either 20 μM or 40 μMmanganese chloride.

6.4. Example 4

6.4.1. Materials & Methods

In the study summarized in this example, we investigated the effects onproduct quality attributes resulting from the addition of manganesechloride and/or galactose to chemically defined Life Technologies GibcoGIA-1 media using a different adalimumab producing CHO cell line than inExamples 1, 2, and 3, named CHO cell line #2.

Growth and production media for the adalimumab-producing CHO cell line#2 were prepared using a proprietary Life Technologies Gibco chemicallydefined media, GIA-1. Production media only was supplemented with withManganese (II) Chloride (Sigma M1787-100 mL; 1.0 M±0.1 M) andD(+)Galactose (Sigma G5388-1 kg) according to the experimental designdescribed in Table 5. All media were filtered through Corning 0.5 L or 1L filter systems (0.22 μm PES) and stored at 4° C. until use.

Upon thaw, cells were expanded for 5 to 8 (2 to 3 day each) passages ina combination of 250 mL, 500 mL, and 1000 mL Corning vented non-baffledshake flasks. Production cultures were initiated in duplicate 500 mLCorning vented non-baffled shake flasks (200 mL working volume) at aninitial VCD of approximately 0.5×106 cells/mL. Cultures were maintainedon orbital shakers at 180 RPM in a dry incubator at 35° C. and 5% CO₂. Aglucose solution (1.25% (v/v) of 40% solution) was fed when the mediaglucose concentration fell below 3 g/L.

6.4.2. Experimental Design

As detailed in Table 5, manganese chloride was supplemented at thefollowing concentrations in production media: 0, 10, 20, and 40 μM.Galactose was supplemented at the following levels in production media:0, 10, and 20 mM. Production media for the control cultures was notsupplemented with manganese chloride or galactose. This study was run in2 blocks.

TABLE 5 Experimental design for Example 4 Manganese Galactose (μM) (mM)ID Block I 0 0 Mn(0) Gal(0) 20 0 Mn(20) Gal(0) 10 10 Mn(10) Gal(10) 2020 Mn(20) Gal(20) 40 20 Mn(40) Gal(20) Block II 0 0 Mn(0) Gal(0) 0 10Mn(0) Gal(10) 0 20 Mn(0) Gal (20) 10 0 Mn(10) Gal(0)

6.4.3. Culture Growth & Productivity

Culture growth, viability profiles, and harvest titer of cultures inproduction CDM GIA-1 supplemented with galactose in the 0 to 20 mMconcentration range and/or manganese chloride in the 0 to 40 μMconcentration range were comparable to the control cultures withoutmanganese or galactose added (FIGS. 13A, 13B; harvest titer data notshown).

6.4.4. Oligosaccharide Analysis

In this example, the modulation of galactosylation by the addition ofmanganese chloride and/or galactose to chemically defined GIA-1 mediawas explored using the adalimumab-producing CHO cell line #2.

The addition of manganese chloride alone within the range of 0 to 20 μMto production CDM GIA-1 decreased the NGA2F+NGA2F-GlcNac sum in aconcentration dependent manner and increased the NA1F+NA2F sum byapproximately the same percentage. A maximum decrease of 22% in theNGA2F+NGA2F-GlcNac sum and a maximum corresponding increase of 23% inthe NA1F+NA2F sum was observed with the addition of 20 μM manganesechloride (FIG. 14). Similarly, a concentration dependent decrease in theNGA2F+NGA2F-GlcNac sum and a corresponding increase in the NA1F+NA2F sumwas observed with the addition of galactose alone in the range of 0 to20 mM. A maximum decrease of 9% in the NGA2F+NGA2F-GlcNac sum and acorresponding maximum increase of 10% in the NA1F+NA2F sum was observedwith the addition of 20 mM galactose.

An additive effect was observed for the oligosaccharide profiles ofadalimumab produced in cultures supplemented with the combined additionof manganese chloride and galactose to GIA-1 media (FIG. 15). Forexample, addition of 10 μM manganese chloride alone reduced theNGA2F+NGA2F-GlcNac sum by 18%, and addition of 10 mM galactose alonedecreased the NGA2F+NGA2F-GlcNac sum by 6%. The combined addition ofmanganese chloride and galactose at these same concentrations led to a24% reduction in the NGA2F+NGA2F-GlcNac sum. The highest percentagedecrease of 35% in the NGA2F+NGA2F-GlcNac sum and the correspondingincrease of 37% in the NA1F+NA2F sum were observed for the culturesupplemented with 40 μM manganese chloride and 20 mM galactose.

6.5. Example 5

6.5.1. Materials & Methods

In the study summarized in this example, we investigated the effects onproduct quality attributes resulting from the addition of manganesechloride and/or galactose to chemically defined Life Technologies GibcoGIA-1 media using a different adalimumab-producing CHO cell line than inExamples 1, 2, 3, and 4, named CHO cell line #3.

Growth and production media for the adalimumab-producing CHO cell line#3 were prepared using the proprietary Life Technologies Gibcochemically defined media, GIA-1. Basal production and feed media weresupplemented with Manganese (II) Chloride (Sigma M3634-100g) andD(+)Galactose (Sigma G5388-1 kg) according to the experimental designdescribed in Table 6.

Upon thaw, cells were expanded in Corning vented non-baffled shakeflasks maintained on orbital shakers at 140 RPM, and in 10 L cell bags(2 L working volume) maintained at 25 RPM, 7° angle, and 0.25 SLPMairflow in a dry incubator at 36° C. and 5% CO₂. Production cultureswere initiated in 3 L bioreactors (1.4 L initial working volume) at 36°C., 30% dissolved oxygen, 200 RPM, and pH 6.9±0.2. A fixed split ratioof cells to media of 1:6.7 was utilized to initiate the production stagecultures. A temperature shift was performed when the culture VCD reacheda value higher than 5×106 cells/mL. The chemically-defined feed fromLife Technologies Gibco JCL-5 was added as follows: 4% (v/v)—day 2,6%—day 4, 8%—day 6, 10%—day 8, and 10%—day 10. Additional 400 g/Lglucose was added to the reactor cultures as needed to ensure glucoselevels did not deplete. Bioreactors were harvested at a viability ofapproximately 50% or on production day 17, whichever condition occurredfirst.

6.5.2. Experimental Design

As detailed in Table 6, manganese chloride was supplemented at thefollowing concentrations in both production and feed media: 0, 0.1, 0.2,0.5, and 1.0 μM. Galactose was supplemented at 0 and 30 mMconcentrations in both production and feed media. In addition, acombined manganese chloride and galactose supplementation strategy wasutilized for the production basal and feed media at either 0.2 or 0.5 μMmanganese chloride plus 30 mM galactose. Basal and feed media for thecontrol cultures were not supplemented with manganese chloride orgalactose.

TABLE 6 Experimental design for Example 5 Manganese Galactose (μM) (mM)ID 0 0 Mn(0) Gal(0) 0.1 0 Mn(0.1) Gal(0) 0.2 0 Mn(0.2) Gal(0) 0.5 0Mn(0.5) Gal(0) 1.0 0 Mn(1.0) Gal(0) 0 30 Mn(0) Gal(30) 0.2 30 Mn(0.2)Gal(30) 0.5 30 Mn(0.5) Gal(30)

6.5.3. Culture Growth & Productivity

Growth profiles of most cultures supplemented with manganese chlorideand/or galactose were comparable to the control culture except for thecultures supplemented with 30 mM galactose alone or in combination with0.2 μM manganese chloride which grew slower and reached a lower peak VCD(FIG. 16A). However, the culture supplemented with 0.5 μM manganesechloride and 30 mM galactose had a growth profile comparable to thecontrol culture indicating that neither manganese chloride nor galactoseat the concentrations studied are detrimental to culture growth.Viability profiles and harvest titer were comparable to the controlcondition (FIGS. 16B, 16C).

6.5.4. Oligosaccharide Analysis

In this example, the modulation of galactosylation with the addition ofmanganese chloride and/or galactose to chemically defined media GIA-1was explored using the adalimumab-producing CHO cell line #3.

The addition of manganese chloride alone within the range of 0 to 1 μMto production CDM GIA-1 decreased the NGA2F+NGA2F-GlcNac sum in aconcentration dependent manner and increased the NA1F+NA2F sum byapproximately the same percentage. A maximum decrease of 26% in theNGA2F+NGA2F-GlcNac and a corresponding increase of 28% in the NA1F+NA2Foligosaccharides were observed with the addition of 1 μM manganesechloride (FIG. 17). The addition of galactose alone at 30 mMconcentration to production CDM GIA-1 decreased the NGA2F+NGA2F-GlcNAcsum by 4% and increased the NA1F+NA2F sum by 3%.

When manganese chloride and galactose were supplemented together intothe production basal and feed media, the results demonstrated asynergistic benefit towards the decrease in the NGA2F+NGA2F-GlcNAc andthe increase in the NA1F+NA2F oligosaccharides which is consistent withthe results demonstrated in Example 1 (FIG. 18). For example, at 0.2 μMmanganese chloride plus 30 mM galactose the observed 25% decrease in theNGA2F+NGA2F-GlcNAc sum was 6% more than the sum of the decrease observedwith the addition of 0.2 μM manganese chloride alone (15%) and that of30 mM galactose alone (4%). Similarly the resulting 24% increase in theNA1F+NA2F sum was more than the sum of the increase observed with theaddition of 0.2 μM manganese chloride alone (16%) and that of 30 mMgalactose alone (3%). The combined supplementation of 0.5 μM manganesechloride+30 mM galactose also demonstrated a synergistic effect on thegalactosylation profile of adalimumab produced in the CHO cell line #3.A maximum decrease compared to the control condition of 34% in theNGA2F+NGA2F-GlcNac and a corresponding 34% maximum increase in theNA1F+NA2F oligosaccharides was observed with the combined addition of0.5 μM manganese chloride and 30 mM galactose to chemically definedGIA-1 media.

6.6. Example 6

6.6.1. Materials & Methods

In the study summarized in this example, we investigated the effects onproduct quality attributes resulting from the addition of manganesechloride and/or galactose to chemically defined Life Technologies GibcoPFBM-3 basal medium and PFFM-4 feed medium (proprietary formulation)using an adalimumab-producing NSO cell line in a fed-batch process inshake flasks.

Growth and production media for the adalimumab-producing NSO cell linewere prepared using a proprietary Life Technologies Gibco chemicallydefined media, PFBM-3 basal medium plus PFFM-4 feed medium. Productionand feed media were supplemented with Manganese (II) Chloride (SigmaM1787-100 mL; 1.0 M±0.1 M) and D(+)Galactose (Sigma G5388-1 kg)according to the experimental design described in Table 7.

Upon thaw, cells were expanded for 3 to 5 (2 days each) passages in acombination of 250 mL, 500 mL, 1 L, 2 L and 3 L Corning ventednon-baffled shake flasks. Production cultures were initiated in single 1L Corning vented non-baffled shake flasks (240 mL initial workingvolume) at an initial VCD of approximately 2.5×105 cells/mL. Cultureswere maintained on orbital shakers at 100 RPM in a dry incubator at 37°C. and 5% CO₂. The shake flask study was run in a fed-batch mode and theculture was fed PFFM-4 as follows: 24 mL—day 2, 28.8 mL—day 4, 28.8mL—day 6, and 28.8 mL—day 8.

Samples were collected every 2 days and measured for cell density andviability using a Cedex cell counter. Retention samples for titeranalysis via Poros A method were collected daily beginning on Day 8 bycentrifugation at 2,000 g for 10 min and then filtered through 0.2 μmPVDF syringe filter. The cultures were harvested on production day 10.The entire culture was collected, chilled on ice to approximately 0° C.for 1.5 hours, the cells and debris flocculated at pH 5.0 by theaddition of 1M citric acid and held for 15 minutes, and centrifuged at4000x g for 15 min at 5° C. The supernatant was passed through 0.20 μmMillipore Stericup PES filters, and, immediately post filtration, theacidified clarified cell-free harvest was neutralized with 2M Tris to pH7.1±0.2. The cell free harvest was transferred to PETG bottles andstored at −80° C. until analysis.

6.6.2. Experimental Design

As detailed in Table 7, manganese chloride was supplemented at thefollowing concentrations in both production and feed media: 0, 0.2, and0.5 μM. Galactose was supplemented at the following levels in bothproduction and feed media: 0, 1, 4, 5, and 10 mM. Manganese chloride andgalactose were added in a full factorial, two level DOE design for the0, 1, and 4 mM galactose conditions and all concentrations of manganesechloride. Individual and combined additions of manganese chloride andgalactose were studied using a comprehensive design divided into 2 setsof experiments. Each experiment had a control culture for directcomparison of culture growth, productivity, and product quality.Production medium for control cultures was not supplemented withmanganese chloride or galactose.

TABLE 7 Experimental design for Example 6 Manganese Galactose (μM) (mM)ID Block I 0 0 Mn(0) Gal(0) 0 5 Mn(0) Gal(5) 0 10 Mn(0) Gal(10) Block II0 0 Mn(0) Gal(0) 0.2 0 Mn(0.2) Gal(0) 0.5 0 Mn(0.5) Gal(0) 0 1 Mn(0)Gal(1) 0.2 1 Mn(0.2) Gal(1) 0.5 1 Mn(0.5) Gal(1) 0 4 Mn(0) Gal(4) 0.2 4Mn(0.2) Gal(4) 0.5 4 Mn(0.5) Gal(4)

6.6.3. Culture Growth & Productivity

Culture growth and viability profiles in production media supplementedwith galactose in the 0 to 5 mM concentration range and/or manganesechloride up to 0.5 μM concentration were comparable to the controlcondition without manganese or galactose added (FIGS. 19A, 19B). Theaddition of galactose at 10 mM concentration had a detrimental effect onculture growth and productivity. The cultures in the Block I experimenthad a lower maximum VCD and overall lower viability than the cultures inthe Block II experiment. All cultures in the Block II experiment showedsimilar VCD and viability profiles. Harvest titer for most experimentalconditions was comparable to the harvest titer for the control conditionexcept for the titer of the culture supplemented with 10 mM galactose,which was 60% lower (FIG. 19C).

6.6.4. Oligosaccharide Analysis

In this example, the modulation of galactosylation by the addition ofmanganese chloride and/or galactose to chemically defined PFBM-3/PFFM-4media was explored using an adalimumab-producing NSO cell line in afed-batch process in shake flasks.

The addition of manganese chloride alone within the range of 0 to 0.5 μMto CDM PFBM-3/PFFM-4 decreased the NGA2F+NGA2F-GlcNac sum in aconcentration dependent manner and increased the NA1F+NA2F sum byapproximately the same percentage. A maximum decrease of 18% in theNGA2F+NGA2F-GlcNac sum and a corresponding increase of 20% in theNA1F+NA2F sum were observed with the addition of 0.5 μM manganesechloride (FIG. 20). Manganese doses greater than 0.5 μM were notexplored further due to cytotoxicity effects. Similarly, a concentrationdependent decrease in the NGA2F+NGA2F-GlcNac sum and a correspondingincrease in the NA1F+NA2F sum were observed with the addition ofgalactose alone in the range of 0 to 10 mM to CDM PFBM-3/PFFM-4. Amaximum decrease of 14% in the NGA2F+NGA2F-GlcNac sum and acorresponding increase of 15% in the NA1F+NA2F sum was observed with theaddition of 10 mM galactose.

The effect on modulation of galactosylation of adalimumab produced in aNSO cell line in CDM PFBM-3/PFFM-4 supplemented with manganese chlorideand galactose was synergistic (FIG. 21). For example, addition of 0.2 μMmanganese chloride alone reduced the NGA2F+NGA2F-GlcNac sum by 12%, andaddition of 4 mM galactose alone decreased the NGA2F+NGA2F-GlcNac sum by2%. However, the combined addition of manganese chloride and galactoseat these same concentrations (i.e. 0.2 μM manganese+4 mM galactose) ledto a 19% reduction in the NGA2F+NGA2F-GlcNac sum, 5% higher than theircombined individual contributions. A maximum decrease of ˜26% in theNGA2F+NGA2F-GlcNac sum and a corresponding 28% increase in the NA1F+NA2Fsum were observed with the combined addition of 0.5 μM manganesechloride and 4 mM galactose.

6.7. Example 7

6.7.1. Materials & Methods

In the study summarized in this example, we investigated the effects onproduct quality attributes resulting from the addition of manganesechloride and/or galactose to chemically defined Life Technologies GibcoGIA-1 media in a CHO cell line producing a monoclonal antibodygenerically named mAb #1.

Growth and production media for the CHO cell line producing mAb #1 wereprepared using a proprietary Life Technologies Gibco chemically definedmedia, GIA-1. Production media only was supplemented with Manganese (II)Chloride (Sigma M1787-100 mL; 1.0 M±0.1 M) and D(+)Galactose (SigmaG5388-1 kg) according to the experimental design described in Table 8.All media were filtered through Corning 0.5 L or 1 L filter systems(0.22 μm PES) and stored at 4° C. until use.

Upon thaw, cells were expanded for 6 (3 day each) passages in acombination of 250 mL, 500 mL, and 1000 mL Corning vented non-baffledshake flasks. Production cultures were initiated in duplicate 500 mLCorning vented non-baffled shake flasks (200 mL working volume) at aninitial VCD of approximately 0.5×106 cells/mL. Cultures were maintainedon orbital shakers at 125 RPM in a dry incubator at 35° C. and 5% CO₂. Aglucose solution (1.25% (v/v) of 40% solution) was fed when the mediaglucose concentration fell below 3 g/L.

6.7.2. Experimental Design

As detailed in Table 8, manganese chloride was supplemented at thefollowing levels in production media: 0, 10, 20, and 40 μM. Galactosewas supplemented at the following levels in production media: 0, 10, 20,and 100 mM. Production media for the control cultures was notsupplemented with manganese chloride or galactose.

TABLE 8 Experimental design for Example 7 Manganese Galactose (μM) (mM)ID 0 0 Mn(0) Gal(0) 0 10 Mn(0) Gal(10) 0 20 Mn(0) Gal(20) 0 40 Mn(0)Gal(40) 10 0 Mn(10) Gal (0) 20 0 Mn(20) Gal(0) 40 0 Mn(40) Gal(0) 10 10Mn(10) Gal(10) 20 20 Mn(20) Gal(20) 40 40 Mn(40) Gal(40)

6.7.3. Culture Growth & Productivity

Cultures supplemented with manganese chloride alone in the concentrationrange of 0 to 20 μM grew comparable to the control cultures (FIG. 22A).Cultures supplemented with galactose alone or with the combination of 20μM manganese and 20 mM galactose grew to a lower maximum VCD compared tothe control, but had the same growth rate until the peak VCD wasachieved on production day 6. These cultures ended a day earlier, onproduction day 9 (FIG. 22B). Cultures supplemented with 40 μM manganesechloride and galactose at all levels studied along with the culturesupplemented with 10 μM manganese chloride and 10 mM galactoseexperienced slower growth and decreased peak VCD compared to thecontrol. Harvest titer was 3-24% lower than the control condition (datanot shown).

6.7.4. Oligosaccharide Analysis

In this example, the modulation of galactosylation by the addition ofmanganese chloride and/or galactose to chemically defined LifeTechnologies Gibco GIA-1 media was explored using a CHO cell lineproducing the monoclonal antibody mAb #1.

The addition of manganese chloride alone within the range of 0 to 40 μMto production CDM GIA-1 decreased the NGA2F+NGA2F-GlcNac sum by amaximum of 26% (FIG. 23). A comparable maximum increase of 27% in theNA1F+NA2F sum was achieved. Addition of galactose alone up to a maximumconcentration of 40 mM yielded a maximum decrease of 12% in theNGA2F+NGA2F-GlcNac sum and a corresponding 13% maximum increase in theNA1F+NA2F sum in a concentration dependent manner.

The combined addition of galactose and manganese chloride to productionCDM GIA-1 resulted in a greater percent reduction in theNGA2F+NGA2F-GlcNac sum and, correspondingly, a greater percent increasein the NA1F+NA2F sum as compared to the addition of either componentalone (FIG. 24). For example, the addition of 40 μM manganese chloridealone reduced the NGA2F+NGA2F-GlcNac sum by 20%, and the addition of 40mM galactose alone decreased the NGA2F+NGA2F-GlcNac sum by 12%. However,the combined addition of manganese chloride and galactose at these sameconcentrations (i.e. 40 μM manganese+40 mM galactose) led to a 27%decrease in the NGA2F+NGA2F-GlcNac sum. The highest percentage decreaseof 32% in the NGA2F+NGA2F-GlcNac sum and the corresponding increase of30% in the NA1F+NA2F sum were observed for the culture supplemented with20 μM manganese chloride and 20 mM galactose.

6.8. Example 8

6.8.1. Materials & Methods

In the study summarized in this example, we investigated the effects onproduct quality attributes resulting from the addition of manganesechloride and/or galactose to chemically defined Life Technologies GibcoGIA-1 media in a CHO cell line producing the monoclonal antibodygenerically named mAb #2 in a fed-batch process in 3 L bioreactors.

Growth and production media for the mAb #2 producing CHO cell line wereprepared using the proprietary Life Technologies Gibco chemicallydefined media, GIA-1. Basal production and feed media were supplementedwith Manganese (II) Chloride (Sigma M3634) and D(+)Galactose (SigmaG5388-1 kg) according to the experimental design described in Table 9.

Upon thaw, cells were expanded in Corning vented non-baffled shakeflasks maintained on orbital shakers at 140 RPM, and in 10 L cell bags(2 L working volume) maintained at 25 RPM, 7° angle, 0.25 SLPM airflowin a dry incubator at 36° C. and 5% CO2. Production cultures wereinitiated in 3 L bioreactors (1.5 L initial working volume) at 36° C.,25% dissolved oxygen, 200 RPM, and pH 7.0. The chemically-defined feedfrom Life Technologies Gibco JCL-5 was added as follows: 3% (v/v)—day 3,5%—day 5, 7%—day 7, 10%—day 9, and 10%—day 11. Additional 400 g/Lglucose was added to the bioreactor cultures as needed to ensure theglucose levels did not deplete. Bioreactors were harvested at viabilityof approximately 70% or below or on production day 15, whichevercondition occurred first.

6.8.2. Experimental Design

As detailed in Table 9, manganese chloride was supplemented at thefollowing concentrations in both production and feed media: 0, 25, 50,and 75 μM. Galactose was supplemented at 0, 15, 30, and 60 mMconcentrations in both production and feed media. In addition, acombined manganese chloride and galactose supplementation strategy wasutilized for the production basal and feed media at 25 μM manganesechloride+15 mM galactose, and 50 μM manganese chloride+30 mM galactose.Basal and feed media for the control cultures were not supplemented withmanganese chloride or galactose.

TABLE 9 Experimental design for Example 8 Manganese Galactose (μM) (mM)ID 0 0 Mn(0) Gal(0) 25 0 Mn(25) Gal(0) 50 0 Mn(50) Gal(0) 75 0 Mn(75)Gal(0) 0 15 Mn(0) Gal(15) 0 30 Mn(0) Gal(30) 0 60 Mn(0) Gal(60) 25 15Mn(25) Gal(15) 50 30 Mn(50) Gal(30)

6.8.3. Culture Growth & Productivity

Growth profiles of most cultures supplemented with galactose in the 0 to60 mM concentration range and/or manganese chloride in the 0 to 75 μMrange were comparable to the control culture except for the culturesupplemented with 25 μM manganese chloride alone which grew slower afterproduction day 7 (FIG. 25A). However, increasing the amount of manganesechloride supplemented to production CDM GIA-1 to 50 μM or 75 μM resultedin cultures with growth profiles comparable to the control culture.Viability profiles and harvest titer were comparable to the controlcondition (FIGS. 25B, 25C).

6.8.4. Oligosaccharide Analysis

In this example, the modulation of galactosylation with the addition ofmanganese chloride and/or galactose to chemically defined media GIA-1was explored using a CHO cell line producing the monoclonal antibody mAb#2.

The addition of manganese chloride alone in the range of 0 to 75 μM toproduction CDM GIA-1 decreased the NGA2F+NGA2F-GlcNAc sum by a maximumof 18% (FIG. 26). A comparable maximum increase of 16% in the NA1F+NA2Fsum was achieved. Addition of galactose alone up to a maximumconcentration of 60 mM yielded a maximum decrease of 12% in theNGA2F+NGA2F-GlcNAc sum and a corresponding 11% maximum increase in theNA1F+NA2F sum.

When manganese chloride and galactose were supplemented together intothe basal and feed media, the results demonstrated at least an additiveeffect and sometimes a synergistic effect towards the decrease in theNGA2F+NGA2F-GlcNAc and the increase in the NA1F+NA2F oligosaccharides(FIG. 27). The synergistic effect was observed for the conditionsupplemented with 25 μM manganese chloride and 15 mM galactose. Theobserved 22% decrease in the NGA2F+NGA2F-GlcNAc sum was 5% more than thesum of the decrease observed with the addition of 25 μM manganesechloride alone (10%) and 15 mM galactose alone (7%). The additive effectwas observed for the condition supplemented with 50 μM manganesechloride and 30 mM galactose. The observed 28% decrease in theNGA2F+NGA2F-GlcNAc sum was comparable to the sum of the decreaseobserved with the addition of 50 μM manganese chloride alone (18%) and30 mM galactose alone (12%). A maximum decrease of 28% in theNGA2F+NGA2F-GlcNAc and a corresponding 25% maximum increase in theNA1F+NA2F sum compared to the control condition was observed with thecombined addition of 50 μM manganese chloride and 30 mM galactose tochemically defined GIA-1 media.

All patents, patent applications, publications, product descriptions andprotocols, cited in this specification are hereby incorporated byreference in their entirety. In case of a conflict in terminology, thepresent disclosure controls.

While it will be apparent that the invention herein described is wellcalculated to achieve the benefits and advantages set forth above, thepresent invention is not to be limited in scope by the specificembodiments described herein. It will be appreciated that the inventionis susceptible to modification, variation and change without departingfrom the spirit thereof.

What is claimed is:
 1. A method for increasing the galactosylation levelof a recombinantly expressed antibody comprising the heavy and lightchain variable domains of adalimumab, comprising supplementing achemically defined (CD) cell culture media used in the expression of theantibody with a manganese supplement, thereby increasing thegalactosylation level of the antibody, wherein the galactosylation levelof the antibody is increased as compared to the galactosylation level ofthe antibody recombinantly expressed in the chemically defined cellculture media which is not supplemented with the manganese supplement.2. The method of claim 1, wherein the antibody is adalimumab.
 3. Themethod of claim 1, wherein the manganese supplement is a biologicallyacceptable manganese salt.
 4. The method of claim 4, wherein thebiologically acceptable manganese salt is manganese (II) chloride. 5.The method of claim 1, wherein the media is supplemented with asufficient amount of the manganese supplement to achieve a manganeseconcentration in the media of 1-100 μM.
 6. The method of claim 1,wherein the media is supplemented with a sufficient amount of themanganese supplement to achieve a manganese concentration in the mediaof 1-40 μM.
 7. The method of claim 1, wherein the media is supplementedwith a sufficient amount of the manganese supplement to achieve amanganese concentration in the media of 40-100 μM.
 8. The method ofclaim 1, wherein the media is supplemented with a sufficient amount ofthe manganese supplement to achieve a manganese concentration in themedia selected from the group consisting of 1.0, 10, 20, 25, 40, 50, 60,75, 80, and 100 μM.
 9. The method of claim 1, wherein the media isfurther supplemented with a galactose supplement.
 10. The method ofclaim 9, wherein the galactose supplement is a biologically acceptablegalactose containing compound.
 11. The method of claim 10, wherein thebiologically acceptable galactose containing compound isD-(+)-galactose.
 12. The method of claim 9, wherein the media issupplemented with a sufficient amount of the galactose supplement toachieve a galactose concentration in the media of 1-100 mM.
 13. Themethod of claim 1, wherein the method further comprises culturing amammalian cell expressing the antibody in the media.
 14. The method ofclaim 13, wherein the mammalian cell has been adapted for growth in a CDcell culture media.
 15. The method of claim 14, wherein the mammaliancell is a CHO cell or an NSO cell.
 16. A process for producing anantibody comprising the heavy and light chain variable domains ofadalimumab, the process comprising culturing a mammalian cell whichproduces the antibody in a chemically defined cell (CD) culture mediathat comprises a concentration of manganese of 1-100 μM, wherein saidconcentration of manganese is sufficient to increase the galactosylationlevel of the antibody as compared to the galactosylation level of theantibody produced in the chemically defined cell culture media that doesnot comprise said concentration of manganese, thereby producing theantibody.
 17. The process of claim 16, wherein the antibody isadalimumab.
 18. The process of claim 16, wherein the media comprises asufficient amount of manganese to achieve a manganese concentration inthe media of 1-40 μM.
 19. The process of claim 16, wherein the mediacomprises a sufficient amount of manganese to achieve a manganeseconcentration in the media of 40-100 μM.
 20. The process of claim 16,wherein the media further comprises galactose.
 21. The process of claim20, wherein the media comprises a sufficient amount of galactose toachieve a galactose concentration in the media of 1-100 mM.
 22. Theprocess of claim 16, wherein the culturing is done in a suspensionculture.
 23. The process of claim 16, wherein the culturing is done in abioreactor.
 24. The process of claim 16, wherein the mammalian cell is aCHO cell or an NSO cell.
 25. The process of claim 16, wherein theprocess is a fed batch process.
 26. The process of claim 16, wherein themedia is selected from the group consisting of production media and feedmedia.
 27. The process of claim 16, further comprising recovering theantibody from the cell culture media.
 28. The process of claim 27,further comprising purifying the antibody from the cell culture media.29. The process of claim 28, further comprising quantifying the levelsof galactose-containing fucosylated biantennary oligosaccharides (NA1Fand NA2F) and/or agalactosyl fucosylated biantennary oligosaccharides(NGA2F and NGA2F-GlcNAc) present on the antibody.
 30. The process ofclaim 17, wherein at least 10% of the total N-linked oligosaccharidespresent on said adalimumab are of a galactose-containing fucosylatedbiantennary oligosaccharide form (sum of NA1F+NA2F).